The role of anti-endothelial cell antibodies in Kawasaki disease - in vitro and in vivo studies

Abstract

Kawasaki disease (KD) is a systemic vasculitis with cardiac and noncardiac complications. Anti--endothelial cell antibodies (AECA) are found among many patients with KD. The aim of this study was to investigate the pathogenic role of AECA in KD using in vitro and in vivo experimental models. F(ab)2 fragments of IgG-AECA and IgM-AECA were affinity purified from a patient with active KD. Their endothelial binding and ability to induce a pro-adhesive and a pro-inflammatory phenotype were evaluated in vitro. Twenty Balb/C mice were immunized with KD-AECA or with control Ig (N-Ig) to induce AECA in a murine model by the idiotypic manipulation method. Both KD-AECA isotypes bind significantly to human umbilical vein endothelial cell (HUVEC) compared to N-Ig. The in vitro activity was demonstrated by the antibodies ability to activate endothelial cells resulting in increased IL-6 secretion, adhesion molecule expression and monocytic cell line (U937) adherence to HUVEC. Five of the mice that received KD-AECA developed murine AECA after 3 months. None of the mice that received N-Ig produced AECA. The murine AECA increased monocyte adhesion to EC in vitro, similarly to the AECA used for immunization. Furthermore, all the mice that developed AECA had proteinuria and IgG deposition in the renal mesangium. No histological or immunofluorescence evidence of cardiac vasculitis could be detected. AECA might play a role in the emergence of some of KD manifestations.

Fig. 1

Binding inhibition of AECA binding to HUVEC. Anti-endothelial cell antibodies from a patient with Kawasaki disease (KD-AECA) at 50% of maximal binding (25 µg/ml) to human umbilical vein endothelial cells (HUVEC) were incubated with increasing concentrations of membrane protein preparations from HUVEC or microvascular EC (human bone marrow EC immortilized by SV-40 [TrHBMEC]). • Takayasu Ig + TrHBMEC; ○ Takayasu Ig +HUVEC; ▪ KD-AECA + TrHBMEC; □ KD-AECA +HUVEC; Takayasu Ig, IgG from a patient with Takayasu arteritis. Values represents the mean ± SD of triplicate measurements from 3 separate experiments.

Fig. 2

Monocyte adhesion to endothelial cell activated by KD-AECA. Adherence of [³H]-thymidine labelled U937 monocytes to human umbilical vein endothelial cells stimulated with increasing concentrations of AECA (○, IgG or ▪ IgM) from a patient with Kawasaki disease (KD) or control (▴ N-IgG; • N-IgM). The results are expressed as percent of added U937 cells that adhered, and are presented as the mean ± SD of triplicate measurements from 3 separate experiments. □ Takayasu-Ig.

Fig. 3

Murine anti-human AECA and murine AECA. Murine anti-human AECA (Ab-2; ▪) and murine AECA (Ab-3; •) in the mice that developed AECA following immunization with affinity purified Ig from a patient with KD-AECA (KD-AECA). Values represent the mean ± SD from 3 separate experiments. ········· Binding of Ab-2 to control-Ig (mean O.D. ± 3 SD). –·–·– Binding of Ig from mice immunized with control-Ig to murine H5V endothelial cells (mean O.D. ± 3 SD).

Fig. 4

Binding inhibition of murine AECA to murine endothelial cells. Inhibition of binding of murine AECA (at 50% of maximal binding- 12·5 µg/ml) to mouse H5V endothelial cells by increasing concentration of HUVEC membranes, but not by human bone marrow microvascular EC immortilized by SV-40 (TrHBMEC) membranes. Values represent the mean ± SD of triplicate measurements from 3 separate experiments. mAECA, Murine AECA from mice immunized with human AECA; N-Ig, IgG from mice immunized with control human IgG. ▪ mAECA + HUVEC, □ mAECA +TrHBMEC, • N-Ig + HUVEC, ○ N-Ig + TrHBMEC.

Fig. 5

Monocyte adhesion to human umbilical vein endothelial cells. Adhesion of [³H]thymidine labelled U937 monocytes to human umbilical vein endothelial cells subjected to mouse AECA (mAECA, ▪) or Ig from mice immunized control (N-Ig, •). Each point represents the mean ± SD of triplicate measurements from 3 separate experiments.

Fig. 6

Immunofluorescent staining of kidney. IgG is detected in the mesangium of mice that developed murine AECA (KD-AECA) (a) and not in those immunized with control Ig (N-Ig) (b). Kidney section (5 µm) stained with FITC-labelled anti-murine IgG ( × 1000).